H2O2 In Endothelial Cells Research Articles

Simultaneous Manipulation and Imaging of Chemogenetically Induced Hydrogen Peroxide in Hardly Transfectable Endothelial Cells

Hydrogen peroxide (H2O2) is a critical signaling molecule in vascular cells, which controls signaling events, yet it can cause pathological oxidative stress in excess. The lack of suitable tools undermined experimental approaches to study the role of oxidative eu- and distress in cellular ultra-locales. This study exploits a yeast-derived D-amino acid oxidase (mDAAO) as a chemogenetic tool to induce, visualize and test the cytotoxicity of H2O2 in hardly transfectable endothelial cells. Due to the poor transfectability of endothelial cells, lentiviral vectors have been used to generate cell lines stably expressing mDAAOs. mDAAOs are substrate-based chemogenetic enzymes that convert D-amino acids to their corresponding alpha-keto acids and generate H2O2 as a byproduct, which can be visualized with a novel ultrasensitive, and ratiometric H2O2 biosensor termed HyPer7. This study tested the suitability of two different D-amino acids, including D-alanine and D-methionine, to induce oxidative stress in endothelial cells. Live-cell imaging experiments unveiled that 10 mM D-methionine generated significantly higher and faster H2O2 signals than D-alanine. However, both D-amino acids induced comparable levels of cell death documented by a colorimetric cell metabolic activity assay (MTT). This study provides a guide for manipulating and monitoring the cytotoxic effect of H2O2 in endothelial cells.

ABCG1 Attenuates Oxidative Stress Induced by H2O2 through the Inhibition of NADPH Oxidase and the Upregulation of Nrf2-Mediated Antioxidant Defense in Endothelial Cells

Summary. Oxidative stress is an important factor that is related to endothelial dysfunction. ATP-binding cassette transporter G1 (ABCG1), a regulator of intracellular cholesterol efflux, has been found to prevent endothelial activation in vessel walls. To explore the role of ABCG1 in oxidative stress production in endothelial cells, HUAECs were exposed to H2O2 and transfected with the specific ABCG1 siRNA or ABCG1 overexpression plasmid. The results showed that overexpression of ABCG1 by ABCG1 plasmid or liver X receptor (LXR) agonist T0901317 treatment inhibited ROS production and MDA content induced by H2O2 in HUAECs. Furthermore, ABCG1 upregulation blunted the activity of prooxidant NADPH oxidase and the expression of Nox4, one of the NADPH oxidase subunits. Moreover, the increased migration of Nrf2 from the cytoplasm to the nucleus and antioxidant HO-1 expression were detected in HUAECs with upregulation of ABCG1. Conversely, ABCG1 downregulation by ABCG1 siRNA increased NADPH oxidase activity and Nox4 expression and abrogated the increase at Nrf2 nuclear protein levels. In addition, intracellular cholesterol load interfered with the balance between NADPH oxidase activity and HO-1 expression. It was suggested that ABCG1 attenuated oxidative stress induced by H2O2 in endothelial cells, which might be involved in the balance between decreased NADPH oxidase activity and increased Nrf2/OH-1 antioxidant defense signaling via its regulation for intracellular cholesterol accumulation.

Abstract 13373: Exercise Improves Angiogenic Function of Circulating Exosomes in Type 2 Diabetes: Role of Extracellular SOD

Background: Exosomes, key mediators of cell-cell communication, derived from type 2 diabetes mellitus (T2DM) have detrimental effects. Exercise not only improves endothelial dysfunction and angiogenesis in T2DM but also induces secretion of exosomes into circulation. Extracellular superoxide dismutase (ecSOD) is a major secretory Cu containing antioxidant enzyme that catalyzes dismutation of O 2 •- to H 2 O 2 and its full activity requires Cu transporter ATP7A. We reported that ecSOD-derived H 2 O 2 in endothelial cells (ECs) enhances angiogenesis while impaired ATP7A-ecSOD axis in diabetes induces endothelial dysfunction. Here we examined whether exercise-derived exosomes (Exe-Exo) may have pro-angiogenic effects via regulating ATP7A-ecSOD axis in T2DM. Results: Two weeks of voluntary wheel exercise of control C57Bl6 mice increased plasma exosome levels (6.2-fold) characterized by Nanosight, TEM and exosome markers (CD63, CD81, Tsg101). Treatment of HUVECs with equal number of exosomes revealed that angiogenic responses such as EC migration (1.8-fold) and tube formation (1.7-fold) were significantly enhanced by Exe-Exo compared to sedentary-derived exosomes (Sed-Exo). This was associated with increased ATP7A (2.9-fold) and ecSOD (1.4-fold) expression in Exe-Exo. Sed-Exo from high fat-induced T2DM mice significantly decreased EC migration (40%) and tube formation (10%) as well as ATP7A expression (28%) compared to Sed-Exo from control mice, which were restored by T2DM Exe-Exo, but not by T2DM/ecSOD KO Exe-Exo. Furthermore, exosomes overexpressing ecSOD (ecSOD-Exo) which mimic exercise increased angiogenesis and H2O2 levels in ECs, which were inhibited by overexpression of catalase. In vivo, skin wound healing model showed that direct application of T2DM Sed-Exo delayed while T2DM Exe-Exo enhanced wound healing of control mice. Furthermore, defective wound healing in T2DM mice or ecSOD KO mice were rescued by ecSOD-Exo application. Conclusion: Exercise training improves pro-angiogenic function of circulating exosomes in T2DM via increasing ATP7A-ecSOD axis, which may provide an effective therapy for promoting angiogenesis and wound repair in metabolic and cardiovascular diseases.

Evolocumab, a PCSK9 inhibitor, protects human endothelial cells against H2O2-induced oxidative stress

Context Recent surveys have shown an association between proprotein convertase subtilisin/kexin type 9 (PCSK9) and oxidative stress. Objective In this investigation, the effect of evolocumab an anti-PCSK9 antibody was assessed against oxidative damage caused by hydrogen peroxide (H2O2) in human umbilical vein endothelial cells (HUVEC). Material and methods Viability of HUVEC was measured by MTT assay. Hydroperoxides and malondialdehyde (MDA) levels, and ferric reducing antioxidant power (FRAP) were detected in HUVEC that pre-treated with evolocumab and, then exposed to H2O2. Results Evolocumab significantly prevented the cytotoxicity induced by H2O2 at the concentrations of 5–100 µg/ml. Pre-treatment of HUVEC with evolocumab reduced hydroperoxides and MDA levels and also increased FRAP value in intra- and extra-cellular mediums compared with H2O2 stimulated cells at different concentration ranges. Conclusion This study displayed anti-oxidative and cytoprotective activities of evolocumab against oxidative damage caused by H2O2 in endothelial cells.

Angiotensin II-induced redox-sensitive SGLT1 and 2 expression promotes high glucose-induced endothelial cell senescence.

High glucose (HG)‐induced endothelial senescence and dysfunction contribute to the increased cardiovascular risk in diabetes. Empagliflozin, a selective sodium glucose co‐transporter2 (SGLT2) inhibitor, reduced the risk of cardiovascular mortality in type 2 diabetic patients but the protective mechanism remains unclear. This study examines the role of SGLT2 in HG‐induced endothelial senescence and dysfunction. Porcine coronary artery cultured endothelial cells (ECs) or segments were exposed to HG (25 mmol/L) before determination of senescence‐associated beta‐galactosidase activity, protein level by Western blot and immunofluorescence staining, mRNA by RT‐PCR, nitric oxide (NO) by electron paramagnetic resonance, oxidative stress using dihydroethidium and glucose uptake using 2‐NBD‐glucose. HG increased ECs senescence markers and oxidative stress, down‐regulated eNOS expression and NO formation, and induced the expression of VCAM‐1, tissue factor, and the local angiotensin system, all these effects were prevented by empagliflozin. Empagliflozin and LX‐4211 (dual SGLT1/2 inhibitor) reduced glucose uptake stimulated by HG and H2O2 in ECs. HG increased SGLT1 and 2 protein levels in cultured ECs and native endothelium. Inhibition of the angiotensin system prevented HG‐induced ECs senescence and SGLT1 and 2 expression. Thus, HG‐induced ECs ageing is driven by the local angiotensin system via the redox‐sensitive up‐regulation of SGLT1 and 2, and, in turn, enhanced glucotoxicity.

Enhancement by Hydrogen Peroxide of Calcium Signals in Endothelial Cells Induced by 5-HT1B and 5-HT2B Receptor Agonists.

Hydrogen peroxide, formed in the endothelium, acts as a factor contributing to the relaxation of blood vessels. The reason for this vasodilatory effect could be modulation by H2O2 of calcium metabolism, since mobilization of calcium ions in endothelial cells is a trigger of endothelium-dependent relaxation. The aim of this work was to investigate the influence of H2O2 on the effects of Ca2+-mobilizing agonists in human umbilical vein endothelial cells (HUVEC). We have found that H2O2 in concentration range 10-100 μM increases the rise of [Ca2+]i induced by 5-hydroxytryptamine (5-HT) and carbachol and does not affect the calcium signals of ATP, agonist of type 1 protease-activated receptor SFLLRN, histamine and bradykinin. Using specific agonists of 5-HT1B and 5-HT2B receptors CGS12066B and BW723C86, we have demonstrated that H2O2 potentiates the effects mediated by these types of 5-HT receptors. Potentiation of the effect of BW723C86 can be produced by the induction of endogenous oxidative stress in HUVEC. We have shown that the activation of 5-HT2B receptor by BW723C86 causes production of reactive oxygen species (ROS). Inhibitor of NADPH oxidases VAS2870 suppressed formation of ROS and partially inhibited [Ca2+]i rise induced by BW723C86. Thus, it can be assumed that vasorelaxation induced by endogenous H2O2 in endothelial cells partially occurs due to the potentiation of the agonist-induced calcium signaling.

Role of E2F transcriptional factor family in hydrogen peroxide regulation of dihydrofolate reductase in endothelial cells (855.5)

Endothelial nitric oxide synthase (eNOS) uncoupling consequent to a deficiency in eNOS cofactor tetrahydrobiopterin (H4B) is an important mechanism of vascular dysfunction. Our previous work has shown that endothelial hydrogen peroxide (H2O2) overproduction specifically downregulates H4B salvage enzyme dihydrofolate reductase (DHFR), which mediates persistent H4B deficiency, eNOS uncoupling and elevated blood pressure in Angiotensin II‐infused mice. However, detailed molecular mechanisms underlying H2O2 regulation of DHFR in endothelial cells remain unclear. In this study, we found that hydrogen peroxide dose and time dependently downregulated DHFR protein expression and its enzyme activity in bovine aortic endothelial cells (BAECs). The DHFR mRNA levels were also decreased by H2O2, implicating transcriptional regulation. Based on the fact that DHFR promoter contains cis‐acting elements for Sp1 and E2F, we constructed wildtype DHFR promoter reporter construct (WT‐DHFR), and mutant DHFR promoter constructs that were deleted of either Sp1 or E2F binding sites. The dual‐luciferase reporter assay showed that while the promoter activities of both WT‐DHFR and Sp1 deletion mutant remained inhibited by H2O2, deletion of the E2F binding sites completely abolished H2O2 attenuation of DHFR promoter activity. RT‐PCR analyses indicated that H2O2 treatment resulted in marked downregulation of E2F1, E2F7 and E2F8 by 38%, 43% and 82% respectively. Intriguingly, overexpression of E2F1, E2F7 or E2F8 restored DHFR expression in response to H2O2, and cotransfection with E2F1 also significantly increased DHFR promoter activity. In conclusion, these results suggest that DHFR is downregulated by H2O2 in endothelial cells transcriptionally via a E2F‐dependent manner, and that restoration of E2F family‐transcription factor activity is effective in preserving DHFR function under oxidative stress.Grant Funding Source: Supported by NHLBI awards HL077440 (HC), HL088975 (HC) and an AHA EI award 12EIA8990025 (HC)

Critical Role of Endothelial Hydrogen Peroxide in Post-Ischemic Neovascularization

BackgroundReactive oxygen species (ROS) play an important role in angiogenesis in endothelial cells (ECs) in vitro and neovascularization in vivo. However, little is known about the role of endogenous vascular hydrogen peroxide (H2O2) in postnatal neovascularization.Methodology/Principal FindingsWe used Tie2-driven endothelial specific catalase transgenic mice (Cat-Tg mice) and hindlimb ischemia model to address the role of endogenous H2O2 in ECs in post-ischemic neovascularization in vivo. Here we show that Cat-Tg mice exhibit significant reduction in intracellular H2O2 in ECs, blood flow recovery, capillary formation, collateral remodeling with larger extent of tissue damage after hindlimb ischemia, as compared to wild-type (WT) littermates. In the early stage of ischemia-induced angiogenesis, Cat-Tg mice show a morphologically disorganized microvasculature. Vascular sprouting and tube elongation are significantly impaired in isolated aorta from Cat-Tg mice. Furthermore, Cat-Tg mice show a decrease in myeloid cell recruitment after hindlimb ischemia. Mechanistically, Cat-Tg mice show significant decrease in eNOS phosphorylation at Ser1177 as well as expression of redox-sensitive vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemotactic protein-1 (MCP-1) in ischemic muscles, which is required for inflammatory cell recruitment to the ischemic tissues. We also observed impaired endothelium-dependent relaxation in resistant vessels from Cat-Tg mice.Conclusions/SignificanceEndogenous ECs-derived H2O2 plays a critical role in reparative neovascularization in response to ischemia by upregulating adhesion molecules and activating eNOS in ECs. Redox-regulation in ECs is a potential therapeutic strategy for angiogenesis-dependent cardiovascular diseases.

Endothelial nitric oxide synthase negatively regulates hydrogen peroxide-stimulated AMP-activated protein kinase in endothelial cells

Hydrogen peroxide and other reactive oxygen species are intimately involved in endothelial cell signaling. In many cell types, the AMP-activated protein kinase (AMPK) has been implicated in the control of metabolic responses, but the role of endothelial cell redox signaling in the modulation of AMPK remains to be completely defined. We used RNA interference and pharmacological methods to establish that H(2)O(2) is a critical activator of AMPK in cultured bovine aortic endothelial cells (BAECs). H(2)O(2) treatment of BAECs rapidly and significantly increases the phosphorylation of AMPK. The EC(50) for H(2)O(2)-promoted phosphorylation of AMPK is 65 + or - 15 microM, within the physiological range of cellular H(2)O(2) concentrations. The Ca(2+)/calmodulin-dependent protein kinase kinase-beta (CaMKKbeta) inhibitor STO-609 abolishes H(2)O(2)-dependent AMPK activation, whereas eNOS inhibitors enhance AMPK activation. Similarly, siRNA-mediated knockdown of CaMKKbeta abrogates AMPK activation, whereas siRNA-mediated knockdown of eNOS leads to a striking increase in AMPK phosphorylation. Cellular imaging studies using the H(2)O(2) biosensor HyPer show that siRNA-mediated eNOS knockdown leads to a marked increase in intracellular H(2)O(2) generation, which is blocked by PEG-catalase. eNOS(-/-) mice show a marked increase in AMPK phosphorylation in liver and lung compared to wild-type mice. Lung endothelial cells from eNOS(-/-) mice also show a significant increase in AMPK phosphorylation. Taken together, these results establish that CaMKKbeta is critically involved in mediating the phosphorylation of AMPK promoted by H(2)O(2) in endothelial cells, and document that eNOS is an important negative regulator of AMPK phosphorylation and intracellular H(2)O(2) generation in endothelial cells.